The present invention relates to a process for transesterifying sterols or stanols in solvent free media and to a process for producing steryl or stanyl esters by enzymatic transesterification of sterols in solvent free media.
Steryl and stanyl esters are useful cholesterol lowering agents and as such are in high demand currently as nutraceutical food ingredients (Food Technology, 2001, 55, 1 pp. 59–67). Steryl and stanyl esters can be made by organic synthesis using different catalysts as disclosed in U.S. Pat. No. 6,184,397. The many disadvantages of organic synthesis is discussed in U.S. Pat. No. 5,219,733 which discloses a process for the enzymatic esterification or transesterification of sterols with fatty acids or with fatty acid esters in an aqueous media or in media containing water saturated organic solvent.
Nevertheless, the disclosed process also suffers from many setbacks. The presence of free water in the reacting mixture may favor hydrolysis over synthesis as can be noticed from the figures shown in the examples, and additionally there are lipases which do not exhibit any activity in water saturated organic solvents. (Yokozeki et al., European J. Appl. Microbiol. Biotechnol., 1982, 14, 1–5). On the other hand, the utilization of organic solvents may not be compatible with the elaboration of nutraceutical food ingredients.
The present invention discloses a novel and high efficiency esterifying process, not only of sterols, but stanols as well, in media without any free water or organic solvent, making the esterifyed products particularly suitable for nutraceutical uses.
In the present invention, it has been observed that certain microbial lipases exhibit transesterifying action on sterols and stanols or on mixtures of these compounds in a reacting system with absence of any organic solvent and free water.
The reacting system is formed when the lipase is contacted with a reactant mixture comprising sterols, stanols, and one or more components selected from the group consisting of fatty acids esterifyed with short chain aliphatic alcohols.
Lipase in the present invention means any formulation expressing transesterifying activity in the absence of free water or organic solvents, and may comprise one or more compounds derived from a fermentation broth of a microorganism or one or more compounds derived from a cellular extract of a microorganism. In these cases the formulation is called free lipase. Alternatively, the formulation may comprise an inert solid immobilizing one or more compounds derived from a fermentation broth of a microorganism or an inert solid immobilizing one or more compounds derived from a cellular extract of a microorganism. In latter cases the formulation is called immobilized lipase. Compounds derived from a fermentation broth or compounds derived from a cellular extract of bacteria of the genus Pseudomonas like Pseudomonas stutzeri or Pseudomonas (Burkholderia) cepacia are suitable for elaborating lipases either free or immobilized.
A lipolytic unit is the amount of formulation that liberates 1 micromole of fatty acid per minute at 37° C. from olive oil emulsified in presence of polyvinyl alcohol. The method for determining lipolytic activity used in the present invention is described in U.S. Pat. No. 5,219,733, being a standard technique for measuring lipolytic activity. It has been observed that there is a correlation between hydrolytic and transesterifying activities of the lipases utilized in the present invention.
The reaction can be carried out in presence either of a stoichiometric amount or stoichometric excess of esters of the fatty acid esters.
Transesterification reaction in the reacting mixture is carried out preferably in agitated reactors at pressures below atmospheric pressure, preferably below 300 mbar and at temperatures ranging from 30 and 90° C.
Fatty acids whose esters are useful for transesterifying sterols or stanols may be derived from an edible oil such as rapseed oil, soybean oil, cottonseed oil, sunflower oil, palm oil, fish oil, and also may comprise fatty acids from 2 to 14 carbon atoms per molecule. These fatty acids can be esterified with a short chain aliphatic alcohol such as methanol, ethanol, propanol or buthanol in presence of sulfuric acid as catalyst, and utilized as transesterifying agents.
Sterols or stanols derived from the residue of the degumming of edible oils such as soy bean oil, sunflower oil, maize germ oil, palm oil, rapeseed oil or the so called wood alcohols which are mixtures of sterols and stanols derived black liquor soaps from Kraft cellulose pulping process, tall oil, or the residue of tall oil distillation known as tall oil pitch, are suitable for the transesterification process disclosed.
In order to finalize the reaction, the lipase is separated from the reacting mixture by either settling, filtering, centrifuging or by any suitable techniques. The resulting reacted mixture comprises steryl or stanyl esters of fatty acids. If stoichiometric excess of esters of fatty acids is used in the transesterifying reaction, the reacted mixture will comprise as well non-reacted excess of these esters. If desired, said excess of esters can be removed from the reacted mixture by distillation at reduced pressure as described in Example 7.
According to what has been disclosed, the process for producing steryl or stanyl esters comprises the steps of:                a) forming a reacting mixture by contacting a lipase with a reactant mixture wherein the reactant mixture comprise sterols or stanols and one or more esters selected from the group consisting of esters of a fatty acid with a short chain aliphatic alcohol;        b) separating the lipase from the reacting mixture to form a reacted mixture; and        c) separating steryl or stanyl esters from the reacted mixture.        
Furthermore, a process for the transesterification of sterols or stanols comprises forming a reacting mixture by contacting a lipase with a reactant mixture wherein the reactant mixture comprise sterols or stanols and one or more esters selected from the group consisting of esters of a fatty acid with a short chain aliphatic alcohol.